Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus, and its recombinant production and activities

Accepted Manuscript Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus, and its recombinant production and activities Hye Young Oh, Chan-Hee Kim, Hye-Jin Go, Nam Gyu Park PII:

S1050-4648(18)30273-0

DOI:

10.1016/j.fsi.2018.05.016

Reference:

YFSIM 5297

To appear in:

Fish and Shellfish Immunology

Received Date: 23 November 2017 Revised Date:

4 May 2018

Accepted Date: 8 May 2018

Please cite this article as: Oh HY, Kim C-H, Go H-J, Park NG, Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus, and its recombinant production and activities, Fish and Shellfish Immunology (2018), doi: 10.1016/j.fsi.2018.05.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Isolation of an invertebrate-type lysozyme from the nephridia of the echiura, Urechis unicinctus,

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and its recombinant production and activities

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Hye Young Oh*, Chan-Hee Kim*, Hye-Jin Go, and Nam Gyu Park†

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Department of Biotechnology, College of Fisheries Sciences, Pukyong National University, 45

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Yongso-ro, Nam-gu, Busan, 48513, Korea

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* These authors contributed equally to this work.

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† Corresponding author: Nam Gyu Park, Department of Biotechnology, College of Fisheries

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Sciences, Pukyong National University, 45 Yongso-ro, Nam-gu, Busan 48513, Korea.

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Tel: +82 51-629-5867

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Fax: +82 51-629-5863

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E-mail: [email protected]

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Keywords: Urechis unicinctus, invertebrate-type lysozyme, protein isolation, recombinant protein

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production, non-enzymatic antibacterial activity

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Abbreviations: AA, amino acid; CFU, colony forming unit; HEWL, hen egg white lysozyme; HPLC,

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high performance liquid chromatography; IPTG, isopropyl β-D-1-thiogalactopyranoside; MEC,

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minimal effective concentration; NAG, N-acetylglucosamine; NAM, N-acetylmuramic acid; nano

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LC-MS/MS, nanoscale liquid chromatography coupled to tandem mass spectrometry; ORF, open

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reading frame; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; Q-TOF, quadrupole

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time-of-flight; RACE, rapid amplification cDNA ends; RT-qPCR, real-time quantitative PCR; SDS-

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PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TSB,

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tryptic soy broth; URDA, ultrasensitive radial diffusion assay; UPLC, ultraperfomance liquid

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chromatography; UTR, untranslated region

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Abstract Invertebrates, unlike vertebrates which have adaptive immune system, rely heavily on the innate immune system for the defense against pathogenic bacteria. Lysozymes, along with other immune

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effectors, are regarded as an important group in this defense. An invertebrate-type (i-type) lysozyme,

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designated Urechis unicinctus invertebrate-type lysozyme, Uu-ilys, has been isolated from nephridia

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of Urechis unicinctus using a series of high performance liquid chromatography (HPLC), and

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ultrasensitive radial diffusion assay (URDA) as a bioassay system. Analyses of the primary structure

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and cDNA cloning revealed that Uu-ilys was approximately 14 kDa and composed of 122 amino acids

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(AAs) of which the precursor had a total of 160 AAs containing a signal peptide of 18 AAs and a pro-

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sequence of 20 AAs encoded by the nucleotide sequence of 714 bp that comprises a 5’ untranslated

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region (UTR) of 42 bp, an open reading frame (ORF) of 483 bp, and a 3’ UTR of 189 bp. Multiple

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sequence alignment showed Uu-ilys has high homology to i-type lysozymes from several annelids.

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Relatively high transcriptional expression levels of Uu-ilys was detected in nephridia, anal vesicle,

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and intestine. The native Uu-ilys exhibited comparable lysozyme enzymatic and antibacterial

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activities to hen egg white lysozyme. Collectively, these data suggest that Uu-ilys, the isolated

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antibacterial protein, plays a role in the immune defense mechanism of U. unicinctus. Recombinant

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Uu-ilys (rUu-ilys) produced in a bacterial expression system showed significantly decreased lysozyme

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lytic activity from that of the native while its potency on radial diffusion assay detecting antibacterial

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activity was retained, which may indicate the non-enzymatic antibacterial capacity of Uu-ilys.

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1. Introduction Echiurans (spoon worms) are a group of marine invertebrates composed of five families:

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Echiuridae, Urechidae, Thalassematidae, Bonelliidae, and Ikedidae [1-3]. Class Echiura had once

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been considered as a separate phylum, however, they are currently regarded as derived annelid

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worms lacking segmentation as suggested by recent molecular phylogenetic studies that classify

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echiurans as a polychaete group in phylum Annelida [4-6]. Most of the echiurans live in burrows in

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soft marine sediments of the lower intertidal and subtidal zones [7-9]. They are generally suspension

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feeders that scoop the sediment with their elongated proboscis to collect small organic particles,

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however, Urechis species is a filter feeder that acquire nutrition from seawater pumped through the

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U-shaped burrow using a mucus net that attaches to the walls of its burrow and gathers food by

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generating water currents using their peristaltic bodies [2, 10-12]. This implies that Urechis species

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require a digestive system capable of degrading or hydrolyzing prokaryotic cells and are in need of

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an effective antimicrobial strategy against harmful organisms for a successful survival in an

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environment that is constituted by approximately 106 bacteria/ml in seawater and 109 bacteria/ml in

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marine sediments [13].

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Antibacterial proteins and peptides generally target essential components of bacteria such as the cell wall, cell membrane, DNA and RNA molecules, and ribosomal subunits. A key group of these

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antibacterial proteins is enzymes (i.e., lysozymes) widely distributed throughout the animal kingdom

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[14-16]. Lysozymes are glycosidases that hydrolyze the 1,4-β-glycosidic linkages between the N-

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acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties that make up peptidoglycan,

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an essential constituent of the bacterial cell wall, and are believed to be involved in digestive

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processes as well as in host defense mechanism [17-20]. From the evolutionary point of view,

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lysozymes are grouped into mainly three distinct categories in the animal kingdom: the chicken (c),

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the goose (g), and the invertebrate (i)-type lysozymes [21, 22]. The phylogenetic distribution of these

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lysozymes revealed that c-type lysozymes are the most conventional one that can be found in a large

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number of vertebrates and in different classes of arthropoda while g-type lysozymes are found in

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several avian species as well as in some bivalve mollusks [21]. Since the first identification of an i-

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type lysozyme from the starfish Asterias rubens [23], many i-type lysozymes have been discovered

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among different invertebrate phyla including mollusks, echinoderms, nematodes, annelids,

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hemichordates, and arthropods [24-32]. I-type lysozymes are approximately 11-13 kDa in size with

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distinct N-terminal amino acid sequences and are characterized by multiple disulfide bonds that are

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highly conserved through the i-type lysozymes from different phyla. Apart from their muramidase

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ACCEPTED MANUSCRIPT activity, that is the hydrolysis of heteropolymer of 1,4-β-glycosidic linked NAM and NAG, most i-

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type lysozymes exhibit chitinase activity (i.e., the hydrolysis of homopolymer 1,4-β-glycosidic linked

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NAG) [21]. Moreover, some i-type lysozymes including Hm-ilys (Hirudo medicinalis, annelids), Vp-

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ilys (Venerupis philippinarum, mollusks), Ea-ilys (Eisenia andrei, annelids), and Pc-ilys2

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(Procambarus clarkii, arthropods) possess isopeptidase activity (i.e., a capacity to split isopeptide

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bond between glutamine γ-carboxamide and ε-lysine amino groups) [28, 30, 33-35]. Moreover, in

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addition to the widely recognized enzymatic activities of i-type lysozymes, a limited number of i-type

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lysozymes, Pc-ilys1 and Aj-ilys (Apostichopus japonicus, echinoderms), are believed to display non-

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enzymatic antibacterial activity [25, 28].

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Urechis unicinctus is an echiuran mainly distributed throughout the coasts of China, Russia, Japan, and Korea. Antimicrobial activity of neuropeptides (urechistachykinins) and hemoglobin (UuHb-F-I)

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had been reported in this species [36, 37]. However, there had not be an adequate investigation on the

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enzymes that are involved in digestion and host defense in Urechis species. In the present study, we

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report the isolation and characterization of an i-type lysozyme (Uu-ilys) from U. unicinctus based on

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antibacterial activity assay. A cDNA encoding the lysozyme was cloned and sequenced, enabling

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investigation of its expression pattern in U. unicinctus. We also describe detection of the enzymatic

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(muramidase) and non-enzymatic activities of the recombinant protein and compare the primary

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structure and activity of Uu-ilys with other i-type lysozymes.

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2. Materials and Methods

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2.1. Animal and sample extraction

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Live specimen of the marine spoon worm, Urechis unicinctus, were purchased from a local fish market in Busan, South Korea. The spoon worms were immediately transferred to our laboratory and

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kept in a recirculating seawater system at 15 oC until sample collection. The nephridia were collected

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from 50 individual animals by cutting and picking up the nephridia at the terminal duct using a knife

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and forceps. Collected sample was immediately frozen and stored at - 75 °C until use. Approval by

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the local institution/ethics committee was not required for this work because the experimental work

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on U. unicinctus is not subject to regulation and U. unicintus is not an endangered or protected species.

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The frozen sample was added to three volumes (w/v) of pre-heated distilled water containing 5%

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acetic acid in a double boiler for 5 min. The boiled sample was cooled on ice and then completely

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homogenized (PT10-35; Kinematica AG, Luzern, Switzerland). The homogenate was then centrifuged

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(13,000 x g, 40 min, 4 °C) and the supernatant was pooled and loaded onto a C18 cartridge (Sep-pak

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C18; Waters Corp). The cartridge was washed with 5% methanol/0.1% trifluoroacetic acid (TFA) and

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retained materials were then eluted with 60% methanol/0.1% TFA. The eluate was concentrated and

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its antibacterial activity against Bacillus subtilis KCTC1021 was evaluated as described below in the

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materials and methods section for antibacterial activity assay.

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2.2. HPLC purification of antibacterial protein

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The 60% methanol eluate on Sep-pak C18 cartridge was applied to a cation-exchange high performance liquid chromatography (HPLC) column (TSK-gel SP-5PW, 7.5 mm × 75 mm, Tosoh Co.,

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Japan). Unbound materials on the column was eluted with 20 mM sodium citrate buffer (pH 5.6) for

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20 min at a flow rate of 1 ml/min, and, then, elution was performed with a linear gradient of 0 to 1.0

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M sodium chloride in 20 mM sodium citrate buffer (pH 5.6) for 50 min at the same flow rate.

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Absorbance was monitored at 220 nm and fractions were collected every 1 min. Fractions responsible

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for strong antibacterial activity, which were eluted between 4 and 10 min with 20 mM sodium citrate

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buffer, were pooled and subjected to a reverse phase HPLC (RP-HPLC) column (CAPCELL-PAK

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C18, 4.6 mm × 250 mm, Shiseido Co., Japan) with a linear gradient of 5 to 65% acetonitrile/0.1%

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TFA for 60 min at a flow rate of 1 ml/min. Fractions were collected every 1 min. Active fractions

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eluted from 29 to 35 min were applied to further purification steps on an anion-exchange column

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(Mono Q HR 5/5, 1 ml, GE Healthcare, USA). Unbound materials on the column was eluted with 20

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mM Tris-HCl buffer (pH 8.0) for 20 min at a flow rate of 1 ml/min and elution was performed with a

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same flow rate and absorbance was monitored at 220 nm. Fractions eluted between 1 and 6 min,

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which were eluted as unbound materials, were pooled and subjected to the RP-HPLC column

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previously used with a linear gradient of 20 to 40% acetonitrile/0.1% TFA for 40 min at a flow rate of

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1 ml/min. Each peak was collected manually. Finally, the peak with antibacterial activity was isolated

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using the same column with a linear gradient of 24 to 30% acetonitrile/0.1% TFA for 30 min at a flow

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rate of 1ml/min. The approximate molecular weight of the purified protein was estimated on 20%

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tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) as described

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previously [38]. An aliquot of each fraction or peak during the purification steps was used to test

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antibacterial activity against B. subtilis KCTC1021.

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2.3. Antibacterial assay

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Ultrasensitive radial diffusion assay (URDA) was employed to measure antibacterial activity as

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previously described [39]. Bacterial strains included the gram-positive bacteria B. subtilis KCTC1021,

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Staphylococcus aureus RN4220, Micrococcus luteus KCTC1071, and Streptococcus iniae FP5229,

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and the gram-negative bacteria Escherichia coli ML35, Salmonella enterica ATCC13311, Aeromonas

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hydrophila KCTC2358, Edwardsiella tarda KCTC12267, and Vibrio anguillarum KCTC2711. Briefly,

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the bacterial strains were pre-grown overnight in tryptic soy broth (TSB) at 37 °C with shaking. Pre-

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cultured bacteria was diluted to concentration of 108 colony forming unit (CFU)/ml with 0.5 of a

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McFarland turbidity standard (Vitek Colorimeter #52-1210; Hach, Loveland, CO, USA) using 20 mM

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phosphate buffer (PB, pH 6.57) with 0.03% TSB (Vitek Colorimeter #52-1210, Hach, USA), and 0.5

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ml of the diluted strains was mixed with 9.5 ml of underlay gel containing 0.03% TSB and 1% Type I

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agarose in 20 mM PB (pH 6.57), followed by transfer to a square petri dish with grids making the

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microbial concentration of the underlay gel into 5 × 106 CFU/ml. Each sample in 5 µl of 0.01% acetic

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acid was added to 2.5 mm diameter wells made in approximately 1 mm thick underlay gel. 0.01%

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acetic acid and 1X PBS were used as negative controls for samples during isolation process and

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recombinant production, respectively. After complete diffusion of each sample for 3 h at room

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temperature, underlay gels containing bacterial strains were overlaid with 10 ml of 6% TSB and 1%

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agarose in 20 mM PB (pH 6.57). The plates were incubated for 16 h, then the diameter of clear zones

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for each sample was measured. The diameter of the well was subtracted, then, the diameter of clear

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zones was expressed in units (0.1 mm = 1 U). Minimal effective concentration (MEC, µg/ml) of the

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tested sample was calculated as the X-intercept of a plot of units against the log10 of the sample

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concentration [40].

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2.4. Primary structure analyses The accurate molecular weight of the intact protein was analyzed using a nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) system with full-scan MS

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mode. The nano LC-MS/MS system was equipped with an Acquity UPLC BEH C18 separation

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column (2.1 mm × 100 mm, 300 Å, 1.7 µm, Waters, Milford, MA, USA) on a nano ultraperfomance

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liquid chromatography (UPLC) system interfaced with quadrupole time-of-flight (Q-TOF) mass

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spectrometer (maXis; Bruker Daltonics, Bremen, Germany) with a nano-electrospray source at

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Cooperation Laboratory Center in Pukyong National University (CLC PKNU). The N-terminal amino

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acid sequence of the protein was analyzed by automated Edman degradation on a pulse liquid

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automatic sequencer (PPSQ-31A/33A protein sequencers, Shimadzu Co., Kyoto, Japan) at CLC

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PKNU.

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2.5. cDNA cloning of the purified antibacterial protein

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Cloning of the cDNA encoding the purified antibacterial protein was performed by 3′ and 5′ rapid amplification of cDNA ends (RACE). Total RNA was extracted from nephridia using Hybrid-R Kit

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(GeneAll Biotechnology, Seoul, Korea) according to manufacturer instructions. Using the total RNA

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and GeneRacer Kit (Invitrogen, CA, USA), RACE-ready cDNA templates were synthesized for

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RACE polymerase chain reaction (PCR) following the manufacturer’s instruction. Based on the

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acquired partial amino acid sequence (AISNNXLSXIXHVEGXERQVGKXRMDRGSL) from N-

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terminal sequencing in which Xs were replaced with cysteine residues, two degenerate primers were

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designed for 3′ RACE (primer sequences used for RACE are listed in Table 1). The first 3′ RACE

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reaction was performed using the degenerate primer Deg-Fw1 and the GeneRacer 3′ primer, and the

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second nested 3′ RACE reaction used the degenerate primer, Deg-nested-Fw2, and the GeneRacer 3′

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nested primer. Both the first and second nested 3’ RACE PCR was performed with following

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conditions: initial denaturation at 95 °C for 2 min, 5 cycles of denaturation at 95 °C for 30 s,

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annealing at 58 °C for 30 s, and extension at 72 °C for 40 s, 5 cycles of denaturation at 95 °C for 30 s,

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annealing at 55 °C for 30 s, and extension at 72 °C for 40 s, 20 cycles of denaturation at 95 °C for 30 s,

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annealing at 52 °C for 30 s, and extension at 72 °C for 40 s, and final extension for 5 min. The

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secondary nested 3′ PCR product was introduced into the pGEM-T easy vector system (Promega,

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Madison, WI, USA) and verified by sequencing. Gene-specific primers (GSP-Rv1 and GSP-Rv2) for

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5′ RACE were designed according to the partial nucleotide sequences acquired from the 3′ RACE

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(Table 1) and were used to amplify 5′ RACE under this condition: initial denaturation at 95 °C for 2

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30 s, 5 cycles of denaturation at 95 °C for 30 s, annealing at 61 °C for 30 s, and extension at 72 °C for

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30 s, 20 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C

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for 30 s, and final extension for 5 min. PCR product of the second nested 5’ RACE-PCR was cloned

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into pTOP TA V2 vector (Enzynomics, Daejeon, Korea), and the sequence was verified by sequencing.

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The full-length cDNA encoding the mature protein was translated into a protein sequence using

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Expert Protein Analysis System (ExPASy) proteomics server of the Swiss Institute of Bioinformatics

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(http://web.expasy.org/translate/) and SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) was used

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to predict the signal peptide of the translated protein sequence. Homology of the translated protein

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was searched through NCBI BLAST and the purified protein was designated as Urechis unicinctus

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invertebrate-type lysozyme (Uu-ilys).

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2.6.Tissue distribution of Uu-ilys transcripts and statistical analysis

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The basal expression levels of the Uu-ilys in five different tissues of U. unicinctus was

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investigated through real-time quantitative PCR (RT-qPCR). The five tissues (i.e., nephridium,

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intestine, hemocyte, body wall, and anal vesicle) were collected from U. unicinctus. Total RNAs were

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extracted from the pooled sample of each tissue (three individuals per pool) using Hybrid-R (GeneAll,

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Seoul, Korea) according to the manufacturer's instructions, and RNA quality was assessed by 1.0%

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agarose gel electrophoresis and then quantified spectrophotometrically using a NanoDrop Lite

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(Thermo Fisher Scientific, Wilmington, MA, USA). cDNA was synthesized from the extracted RNA

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using the TOPscript cDNA synthesis Kit with oligo dT (dT18) (Enzynomics, Daejeon, Korea)

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according to the manufacturer's instructions. The primer pairs designated as Uu-ilys qPCR-F and

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qPCR-R were used for amplifying Uu-ilys cDNA (GenBank accession no. MG372494). U. unicinctus

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β-actin (GenBank accession no. GU592178) was used as an internal control for normalization and the

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primer pairs β-actin qPCR-F and qPCR-R were used for amplification of β-actin cDNA as described

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previously (see Table 1 for sequences) [41, 42]. To quantitatively analyze expression of Uu-ilys

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transcripts in different spoon worm tissues/organs, RT-qPCR was employed using a CFX Connect

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Real-Time PCR Detection System (Bio-Rad, USA) as previously described with slight modification

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[43]. In brief, the amplification was carried out in 20 µl volume reaction mixture containing 10 µl of

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2× SYBR green premix (TOPreal qPCR 2X PreMix, Enzynomics, Daejeon, Korea), 1 µl (10 pmol/µl)

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of each forward and reverse primers, 1 µl of 10-fold diluted cDNA templates, and then nuclease-free

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water was added to make the final volume 20 µl. The thermal profile was 95 °C for 10 min, 40 cycles

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of 95 °C for 10 s, 60 °C for 15 s and 72 °C for 15 s with fluorescence recording at the end of each

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cycle. Melt curve analysis was performed to ensure product specificity over the temperature range of

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60-90 °C. Amplicons were analyzed on agarose gels to confirm product size. Based on the standard

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curves of both Uu-ilys and β-actin, the relative expression levels of the Uu-ilys transcripts in each

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tissue were normalized against the level of the β-actin control using the comparative CT method (2-

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∆∆CT

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analyzed. For statistical analysis of Uu-ilys transcript expression, the graphs were generated, and one-

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way analysis of variance (ANOVA) with Bonferroni multiple range post-hoc analysis was performed

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using GraphPad Prism software version 7.0 for Windows (GraphPad Software, San Diego, California,

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USA). Relative fold expression was presented as means ± standard deviation. P values with p < 0.05

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were considered statistically significant.

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2.7.Production of the recombinant Uu-ilys (rUu-ilys)

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The nucleotide sequence encoding rUu-ilys was amplified by PCR using the forward primer Uu-

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) [44]. Triplicate amplifications were carried out independently, and the results were statistically

ilys-BamHI-Fw and the reverse primer Uu-ilys-XhoI-Rv (primer sequences are listed in Table 1). The

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PCR product was cloned in-frame using BamHI/XhoI site of pET-28a(+) vector and verified by

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sequencing. The constructed pET-28a-Uu-ilys plasmid was transformed into E. coli BL21 (DE3) cells

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(Novagen, Madison, WI, USA) for expression of rUu-ilys. The single colony of transformed cells was

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pre-cultured overnight at 37 °C in Luria-Bertani (LB) broth supplemented with 30 µg/ml kanamycin.

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The pre-cultured cells were inoculated into 500 ml of LB broth supplemented with 30 µg/ml

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kanamycin, and, then the cells were grown at 37 °C to 0.6 of an optical density at 600 nm. Expression

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of rUu-ilys was induced with 0.25 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 6 h

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and bacterial cells were harvested by centrifugation. The cells were washed with 1× phosphate-

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buffered saline (PBS; pH 7.4) three times and lysed by resuspending bacterial pellets in 1× PBS,

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followed by three sonication steps at 40% amplitude for 20 s using a Sonifier 250 (Branson Ultrasons,

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Annemasse, France). The lysate was centrifuged (20,000 x g, 20 min, 4 °C), and the precipitate was

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dissolved in 1× PBS containing 8 M urea and 5 mM imidazole prior to His-tag-affinity purification.

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His-tagged Uu-ilys protein was purified using affinity chromatography by incubating the resuspended

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precipitate with nickel-nitrilotriacetic acid resin (Novagen) at a ratio of 30:1 (v/v) for 1 h at 25 °C and

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then eluted with four column volumes of 1× PBS (pH 7.4) containing 8 M urea and 500 mM

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imidazole. The affinity purified rUu-ilys protein in 8 M urea was then dialyzed using 7 steps of

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dialysis. Each step was conducted with declining stepwise urea concentration: 8, 6, 4, 2, 1, 0.5, 0 M

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urea in 1X PBS. Each dialysis step was performed at 4 °C for at least 8 h.

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2.8.Lysozyme activity assay Lysozyme activity was assessed by measuring the lysis of the Micrococcus lysodeikticus cells

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using lysozyme activity kit (Sigma-Aldrich, USA) according to the manufacturer instructions. Briefly,

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800 µl of M. lysodeikticus bacterial suspension was mixed with 30 µl of lysozyme solution in a

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cuvette. M. lysodeikticus bacterial suspension, which was used as the substrate for lysozyme activity,

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was prepared in the reaction buffer of 66 mM potassium phosphate (pH 6.24) with absorbance at 450

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nm between 0.6-0.8. Lysozyme solution was prepared immediately before use. The concentration of

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lysozyme solution of native Uu-ilys and rUu-ilys was determined by using a calibration curve

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generated by a range of 0 to 1 mg/ml concentration of hen egg white lysozyme (HEWL) solution as a

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standard through running an SDS-PAGE. Immediately after lysozyme solutions were mixed with

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substrate solution, absorbance at 450 nm was measured for 60 min. Absorbance was measured every

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minute for 5 min and then every 10 min until 60 min. All assays were performed at room temperature.

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The activity of each lysozyme is shown in percent lysis of M. lysodeikticus determined with

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absorbance at 450 nm. The absorbance at 0 min was considered 0% lysis.

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% lysis = [1 – (A450 at each time point / A450 at 0 min)]·100% 2.9. in silico analysis

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2.9.1. Multiple sequence alignment and phylogenetic analyses of Uu-ilys

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Multiple sequence alignment of Uu-ilys and other i-type lysozymes was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) and refined manually in Bioedit

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(http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Seven i-type lysozymes from H. medicinalis

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(phylum Annelida, gb|AAA96144|) [45], V. philippinarum (phylum Mollusca, Swiss-Prot |Q8IU26|)

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[46], Caenorhabditis elegans (phylum Nematoda, gb|CCD64578|) [47], Drosophila melanogaster

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(phylum Arthropoda, gb|AAL49382|), Suberites domuncula (phylum Porifera, gb|CAG27844|) [48], A.

278

japonicus (phylum Echinodermata, gb|ABK34500|) [25], and Branchiostoma japonicum (phylum

279

Chordata, gb|AHJ11174|) were aligned with Uu-ilys. The phylogenetic analysis was conducted using

280

Mega 7.0 program and a consensus tree was then constructed by Neighbour-Joining (NJ) method [49].

281

The robustness of each topology was checked by 1000 bootstrap replications. The amino acid

282

sequences of i-type lysozymes from different organisms used for phylogenetic analysis were obtained

283

from the GenBank database. The 3D structure of Uu-ilys was predicted by homology modeling using

284

the structure of Vp-ilys from V. pillippinarum (PDB|2DQA.1A|) as a template in the SWISS-MODEL

285

server (https://swissmodel.expasy.org/).

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2.9.2. Prediction of regions in Uu-ilys for non-enzymatic antibacterial activity To predict the non-enzymatic antibacterial activity of regions in Uu-ilys, the amino acid sequence of Uu-ilys was roughly fragmented into several regions reflecting its secondary structural features.

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The antibacterial properties of each fragment were assessed using antimicrobial peptide (AMP)

290

prediction tools provided by three different databases: Antimicrobial Peptide Database (APD,

291

http://aps.unmc.edu/AP/prediction/prediction_main.php), Collection of Anti-Microbial Peptides

292

(CAMP, http://www.camp.bicnirrh.res.in/prediction.php), Database of Antimicrobial Activity and

293

Structure of Peptides (DBAASP, https://dbaasp.org/prediction). The total hydrophobicity ratio, net

294

charge, grand average of hydropathicity index (GRAVY), Wimley-White hydrophobicity (Kcal/mol),

295

and helical formation data were obtained from APD. AMP probability using Random Forest classifier

296

and AMP prediction were obtained through CAMP and DBAASP, respectively.

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3. Results

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3.1 Isolation of the antibacterial protein To purify antibacterial proteins from nephridia extract of U. unicinctus, 60% methanol/0.1% TFA

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eluate on Sep-Pak C18 cartridge, which exhibited potent antibacterial activity against B. subtilis, was

301

used as the source of isolation. B. subtilis was used for checking antibacterial activity in the

302

subsequent purification steps. The 60% methanol eluate was firstly applied to a cation-exchange

303

HPLC, then eluted with sodium chloride. Unbound materials eluted between 4 to 10 min showed

304

antibacterial activity against B. subtilis (Fig. 1A). These materials were pooled and separated further

305

through RP-HPLC (Fig. 1B). Active materials eluted from 25 to 32% acetonitrile/0.1% TFA had

306

antibacterial activity and then was separated by anion exchange HPLC (Fig. 1C). Unbound fractions

307

on anionic-exchange HPLC eluted between 1 to 6 min exhibited antibacterial activity and was further

308

purified using RP-HPLC (Fig. 1D). Finally, a single absorbance peak with antibacterial activity was

309

eluted at 26-27% acetonitrile/0.1% TFA on RP-HPLC (Fig. 2A).

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3.2 Structural analyses of the purified protein

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The purified protein on the final step of RP-HPLC migrated to approximately 14 kDa on 20%

312

tricine-SDS-PAGE, which was consistent with the molecular weight of the purified intact protein

313

determined to be 13295.1 Da by LC-MS (Fig. 2B, C). Edman degradation method was used to acquire

314

N-terminal amino acid sequence, which gave a partial sequence of 30 AAs in the N-terminus of the

315

isolated protein: AISNNXLAXIXQVEGXESQVGKXRMDRGDL (Fig. 2D). NCBI BLAST search

316

for homology using the acquired partial amino acid sequence revealed that this protein was highly

317

similar to i-type lysozymes described in other annelid species: 66% identities with E. andrei

318

(gb|ABC68610|) and Eisenia fetida (gb|AGJ83864|) and 62% with H. medicinalis (gb|AAA96144|)

319

(Fig. 2D). Based on the homology search results, the isolated protein was assumed to be an i-type

320

lysozyme from U. unicinctus and was named Urechis unicinctus-invertebrate type lysozyme (Uu-ilys).

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3.3 cDNA cloning of Uu-ilys

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cDNA encoding Uu-ilys was determined through 3’ and 5’ RACE PCR. The full nucleotide

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sequence comprises 714 bp including a 5’ untranslated region (UTR) of 42 bp, an open reading frame

324

(ORF) of 483 bp, and a 3’ UTR of 189 bp containing a polyadenylation consensus sequence

325

(AATAAA) located 11 bp upstream of the poly(A)+ tail. The ORF was translated into an amino acid

326

sequence containing 160 AAs. It had a signal peptide of 18 AAs determined using SignalP and a pro12

ACCEPTED MANUSCRIPT sequence of 20 AAs and a mature form of 122 AAs determined based on N-terminal sequencing data

328

(Fig. 3). The translated mature Uu-ilys had a calculated molecular weight of approximately 13.5 kDa,

329

which was consistent with the results of tricine-SDS-PAGE (Fig. 2B). However, it was slighted larger

330

than 13.3 kDa determined by LC-MS. Collectively, Uu-ilys is a protein produced from a precursor

331

that possesses pre-pro-sequence. It’s molecular weight, in accordance with data obtained from

332

primary structure determination and cDNA cloning, was approximately 13.3 kDa. The differences in

333

molecular weight of Uu-ilys suggest presence of C-terminal deletion or other post-translational

334

modifications.

335

3.4 Tissue distribution of Uu-ilys transcripts

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Distribution of Uu-ilys precursor transcripts in five different tissues of U. unicinctus (nephridium,

337

intestine, hemocyte, body wall, and anal vesicle) was determined using U. unicinctus β-actin gene, an

338

adequate housekeeping gene, as an invariant control for the comparison of relative expression

339

between transcripts (Fig. 4). Highest expression of Uu-ilys-precursor transcripts was detected in the

340

nephridia (p <0.05). Additionally, relatively high transcriptional expression levels were observed in

341

anal vesicles and intestine while transcript expression levels in body wall and hemocyte were low (p

342

<0.05).

343

3.5 Recombinant protein (rUu-ilys) production and lysozyme and antibacterial activities

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recombinant Uu-ilys (rUu-ilys) in pET-28a(+) vector was overexpressed in Escherichia coli BL21

346

(DE3) by 0.25 mM IPTG at 37 °C for 6 h as insoluble protein (Fig. 5A, B). The insoluble proteins

347

were solubilized in 8 M urea then purified using His-tag affinity chromatography. However, the

348

purified protein precipitated when dialyzed against 1X PBS. A stepwise dialysis was utilized to

349

promote proper refolding of the recombinant protein. The dialysis solubilized the insoluble rUu-ilys,

350

which was then concentrated for further use. Approximately 1.5 mg of rUu-ilys was produced from E.

351

coli culture of 1.5 liters. The purified rUu-ilys was compared with its native form on SDS-PAGE (Fig.

352

5C). rUu-ilys migrated to between 15 kDa and 20 kDa on a 15% polyacrylamide gel while Uu-ilys

353

migrated to below 15 kDa due to the mass difference caused by His-tag attachment to rUu-ilys.and

354

mass difference observed between cDNA cloning and LC-MS data.

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3.6 Muramidase and antibacterial activities

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rUu-ilys was utilized in the lysozyme enzymatic (muramidase) and antibacterial activities against 13

ACCEPTED MANUSCRIPT various strains compared with the native and HEWL. The muramidase activity of rUu-ilys compared

358

with those of native Uu-ilys and HEWL was significantly weaker, almost undetected when same

359

concentration (0.5 µg/ml) of each sample was used (Fig. 6A). Percent lysis of M. lydeikticus (% lysis)

360

at 5 min for rUu-ilys (0.5 µg/ml) was only 1.17% while same concentrations of Uu-ilys and HEWL

361

were able to lyse 38.58% and 31.24%, respectively. Moreover, 20-fold concentration of rUu-ilys (10

362

µg/ml) lysed 4.3% of M. lydeikticus suspension at 5 min, which was merely 11.1% and 13.8% of Uu-

363

ilys and HEWL activity, respectively. At 60 min, rUu-ilys (0.5 µg/ml) was only able to break down

364

9.68% of M. lydeikticus while bacterial suspension treated with Uu-ilys and HEWL had 77.83% and

365

95.93% lysis, respectively. Interestingly, 20-fold concentration of rUu-ilys (10 µg/ml) lysed

366

approximately half of M. lydeikticus (47.61%), which was 61.2% and 49.6% of Uu-ilys and HEWL

367

activity at 60 min. Collectively, rUu-ilys exerted much weaker lysis activity than Uu-ilys and HEWL

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at the same concentration suggesting a significant decrease in muramidase activity. This decrease in

369

enzymatic antibacterial activity of rUu-ilys may be caused by the failure of proper folding.

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Contrary to decreased enzymatic activity, rUu-ilys exhibited comparable antibacterial activity to that of the native. Among the four gram-positive bacteria including a fish pathogen and five gram-

372

negative bacteria including three fish pathogens, B. subtilis, M. luteus, S. enterica, and E. coli were

373

most susceptible to the tested lysozymes: Uu-ilys and rUu-ilys, which was compared to HEWL at the

374

same concentration (100 µg/ml) (Fig. 6B). The MEC (µg/ml) for antibacterial activity of Uu-ilys, rUu-

375

ilys, and HEWL against the four most susceptible strains were measured (Fig. 6C). Uu-ilys was most

376

potent against B. subtilis (0.07 µg/ml) and S. enterica (0.52 µg/ml). While similar pattern emerged in

377

rUu-ilys antibacterial activity, which had MEC values of 1.03 µg/ml and 2.93 µg/ml against B. subtilis

378

and S. enterica, HEWL was most potent against Gram-positive bacteria, B. subtilis (0.26 µg/ml) and

379

M. luteus (1.77 µg/ml). Collectively, despite decreased lysozyme enzymatic activity, rUu-ilys

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exhibited antibacterial activity that was comparable to the native Uu-ilys indicating an alternative

381

antibacterial mechanism exists.

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3.7 Alignment and phylogenetic analysis of Uu-ilys

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Homology search using the full amino acid sequence (160 AAs) of Uu-ilys revealed that this

384

protein is similar to i-type lysozymes described in various invertebrate phyla (Annelida, Mollusca,

385

Nematoda, Arthropoda, Porifera, Echinodermata, and Chordata) (Fig. 7A) [14, 25, 26, 34, 35, 50].

386

The alignment showed conservation of a few sequences that are deemed important for i-type

387

lysozyme activity. The catalytic residues, Glu14 and Asp26 (residue number taken from the sequence of 14

ACCEPTED MANUSCRIPT Uu-ilys mature form), for the muramidase activity were conserved in all the i-type lysozymes used in

389

this alignment except for the i-type lysozyme from D. melanogaster. The i-type lysozymes aligned

390

had conserved catalytic residues, Ser62 and His92, for isopeptidase activity. Additionally, lysozymes

391

also shared the glycoside hydrolase motif (30LSCGPFQIK38) and multiple cysteine residues (Fig. 7A).

392

Interestingly, the secondary structures of Vp-ilys from V. philippinarum (PDB|2DQA.1A|) mostly

393

coincide with the conserved regions of i-type lysozyme sequences. Phylogenetic tree was built with

394

the protein sequences of several i-type lysozymes using ML method to evaluate the relationships

395

among the i-type lysozymes (Fig. 7B). Uu-ilys clustered in the annelid i-type lysozyme group, which

396

contain Urechis species, that closely clustered with mollusk group. The arthropod i-type lysozyme

397

group was clustered furthest away from the annelid group suggesting that arthropod i-type lysozymes

398

are most different from Uu-ilys among the studied i-type lysozymes.

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ACCEPTED MANUSCRIPT 399

4. Discussion Marine filter feeders like U. unicinctus acquire nutrition from microbe-rich seawater and require

401

an effective antibacterial strategy against harmful organisms [2, 13, 51]. Filter feeding invertebrates

402

rely heavily on the innate immune system, which contain antibacterial proteins like lysozymes [21].

403

This study reports the isolation of an antibacterial protein, Uu-ilys, from nephridia of U. unicinctus,

404

which possessed characteristic features of i-type lysozymes (i.e. high cysteine content, glycoside

405

hydrolase motif, and the catalytic amino acid residues). Nephridia was the source of protein isolation

406

and high transcriptional expression suggesting this tissue’s involvement in the immune defense

407

mechanism of U. unicinctus. The recombinant Uu-ilys shows slightly different activity profile; rUu-

408

ilys was not able to exert enzymatic lytic activity to the full capacity shown by native Uu-ilys,

409

however, its antibacterial activity was comparable to that of the native suggesting a non-enzymatic

410

antibacterial mechanism of Uu-ilys may exist.

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Uu-ilys is an i-type lysozyme with structural features that are conserved among the majority of itype lysozymes described in different phyla (Mollusca, Nematoda, Arthropoda, porifera,

413

Echinodermata, and Chordata), namely, the glycoside hydrolase motif, catalytic residues, and multiple

414

cysteines [14, 22, 52]. The glycoside hydrolase motif in lysozymes is a shared common structural

415

motif (30LSCGP/YFQIK38) that forms a β-hairpin [14, 52]. This motif is shared in all the glycoside

416

hydrolase family, which includes lysozymes and chitinases, and is characterized by a β–hairpin with a

417

type I β-turn (β2-3) near the catalytic residues in the N-terminal region and is positioned spatially

418

close to the substrate binding site [48]. Along with the four catalytic residues for muramidase and

419

isopeptidase activities that are mostly conserved among the i-type lysozymes, several residues are

420

reported to be potentially involved in the lysozyme-substrate interaction in Vp-ilys [14, 53]. The high

421

content of cysteines, which are likely to form multiple disulfide bonds which then, in turn, would

422

contribute to the structural stability, distinguishes i-type lysozymes from other types of lysozymes

423

such as c-type, g-type, and ch-type lysozymes [14, 32]. In the context of conservation of structural

424

features and biological activities of lysozymes, proper folding of lysozyme structure seems important

425

in exerting lysozyme activities. Vp-ilys, an i-type lysozyme described in V. philippinarum, is reported

426

to resist more to denaturation conditions (e.g. heat, 8M urea, etc.) than HEWL and human c-type

427

lysozyme [32]. Consistently, Uu-ilys was isolated from the boiled extract of nephridia. However, it

428

has been reported that a long period of exposure to heat may degrade the i-type lysozyme’s proper

429

folding and cause decrease or loss of enzymatic antibacterial activity [54].

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Uu-ilys was isolated from U. unicinctus, which is a filter-feeding annelid that lives in a soft marine sediment [2]. This animal filters through sea water for nutrients and is in need of an effective

432

immune system to protect itself from the microbes that pass through the animal [8, 10, 55]. Although

433

nephridia in Urechis seem to function more as a storage organ for gametes than excretory organ,

434

nephridia and anal vesicles are regarded as organs responsible for excretion of wastes, which could

435

have constant contact with microbes [51]. Additionally, nephridia were the source of the antibacterial

436

protein, Uu-ilys, isolation and its high transcriptional expression level, which suggest that nephridia is

437

involved in the immune defense mechanism of U. unicinctus. The ubiquitous expression of the

438

isolated protein, Uu-ilys, in five tissues of U. unicinctus determined by RT-qPCR suggests Uu-ilys

439

could be essential in the immune system of this animal. However, since the highest expression level

440

was observed in nephridia it might be possible for Uu-ilys to be produced in nephridia then

441

transported to other tissues of this animal. Relatively high transcriptional expression levels were also

442

detected in anal vesicle and intestine. Nephridia and anal vesicles are excretory organs and intestine is

443

reported to hold filtrating function in U. unicinctus [55]. These organs have high contact with

444

microbes that pass through U. unicinctus and, thus, could play an important role in the immune

445

defense against microbes.

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The recombinant protein (rUu-ilys) exhibited much weaker enzymatic activity compared to its

447

native form. Due to the significant decrease in lysozyme enzymatic antibacterial activity, rUu-ilys

448

was predicted to exert weak antibacterial activity against the tested microbes. On the contrary, rUu-

449

ilys had potent antibacterial activity against B. subtilis, S. enterica, and E. coli. This antibacterial

450

activity could be due to the non-enzymatic antibacterial activity of lysozymes. There have been a few

451

reports of non-enzymatic antibacterial activity of lysozymes, in which it is suggested that non-

452

enzymatic antibacterial activity could be due to lysozymes’ cationic polypeptide regions that act as

453

antimicrobial peptides against microbes [21, 25, 50, 54]. The cationic region of the lysozymes could

454

interact with the negatively charged bacterial cell envelope eventually stopping bacterial growth or

455

killing bacteria [56, 57]. Moreover, two synthesized fragments of an i-type lysozyme from H.

456

medicinalis with lengths of 11 AAs and 8 AAs are reported to exert antibacterial activity [54].

457

Interestingly, rUu-ilys had relatively weak antibacterial activity against gram-positive bacterium, M.

458

luteus while showing potent antibacterial activity against B. subtilis, which could be due to the

459

different composition and modifications in bacterial cell wall [58].

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The multiple sequence alignment of Uu-ilys and i-type lysozymes from different invertebrate phyla revealed that the conservation of amino acid residues occurred mostly in the secondary 17

ACCEPTED MANUSCRIPT structures of Vp-ilys from V. pillipparum suggesting the secondary and tertiary structures in the i-type

463

lysozymes are conserved. The 3D structure of Uu-ilys was predicted by homology modeling using the

464

structure of Vp-ilys (2DQA.1A) as a template in the SWISS-MODEL server (Fig. 8A) [53]. The 3D

465

model of Uu-ilys, just like other i-type lysozymes, contains the glycoside hydrolase motif (α-helix 1

466

and β-strands 2 and 3) and the catalytic residues responsible for muramidase activity (Glu14 and Asp26)

467

and isopeptidase activity (Ser62 and His92) [14, 21]. β-strand 3 is absent in the 3D model of Uu-ilys,

468

however, β-strand 3 in the template structure was also very short and was composed merely of two

469

amino acids, Ile36 and Lys37. These two amino acid residues were conserved in all i-type lysozymes

470

used in the multiple sequence alignment, suggesting the conservation of β-strand 3 (Fig. 7A).

To predict antibacterial regions in Uu-ilys, the amino acid sequence of Uu-ilys was roughly

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fragmented into seven regions reflecting the secondary structural features: α1, β1-3, α2, α3-4, α5, α6,

473

α5-6 (Fig. 8B). AMP prediction of these regions conducted using parameters for AMPs from three

474

databases (APD, CAMP, and DBAASP) revealed that the region containing α-helices 5 and 6 (α5-6) is

475

most likely to exert antibacterial activity while the region contacting α-helix 2 (α2) is the least likely

476

to act as an AMP (Table 2). The parameters for the majority of AMPs are a net charge between -5 and

477

+10, a hydrophobicity between 10% and 80% (peak distribution between 40% and 50%), and a length

478

between 5-60 amino acids [59]. α5-6 region is composed of 37 AAs with total hydrophobicity of 32%

479

and net charge of +3. The region containing α-helices 3 and 4 (α3-4), which is composed of 19 AAs

480

with hydrophobicity of 57% and net charge of +2, also has a high possibility of possessing

481

antibacterial activity. It has been reported that the synthesized peptide composed of amino acid

482

residues that form α-helix 4 in the i-type lysozyme from H. medicinalis (HAYMDRYARRC)

483

possesses powerful antibacterial activity while peptides containing α-helix 5 (CQDYAKIH) or α-helix

484

6 (YWDNVRRC) were inactive or active only against M. luteus [54]. Moreover, helical wheel

485

projections of α-helices 5 and 6 show both structures have amphipathic properties and are cationic

486

(Fig. 8C and D). α-helix 5 has hydrophobic residues (Ala89, Ile91, and Cys99) on one side while

487

charged residues (positive, Lys90, Lys101, and Arg97; negative, Glu86 and Asp87) cluster on the other

488

side. Similarly, α-helix 6 has multiple hydrophobic residues (Leu103, Trp107, Val110, Cys113, and Cys114)

489

on one side and charged residues (positive, Arg108, Arg109, and Lys111; negative, Glu104 and Asp115) on

490

the other. Many AMPs reported thus far form amphipathic structures and are often cationic at

491

physiological pH [60]. Taken together, Uu-ilys region containing α-helices 5 and 6 at the C-terminus

492

is most likely to exert antibacterial activity and have helical formation contributing to non-enzymatic

493

antibacterial activity of Uu-ilys.

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2017-0366).

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This work was supported by a Research Grant of Pukyong National University in 2017 (C-D-

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Acknowledgment

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lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme

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Barsova, E.D. Sverdlov, Genes from the medicinal leech (Hirudo medicinalis) coding for unusual

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enzymes that specifically cleave endo-epsilon (gamma-Glu)-Lys isopeptide bonds and help to dissolve

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blood clots, Mol. Gen. Genet. 253(1-2) (1996) 20-25.

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complete cDNA sequence, construction of expression systems, and elucidation of fibrinolytic activity

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for Tapes japonica lysozyme, Protein Expr. Purif. 36(2) (2004) 254-262.

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induction of lysozyme and AdaPTin, Mar. Biol. 146(2) (2005) 271-282.

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shrimp, Litopenaeus vannamei, Fish Shellfish Immunol. 54(Supplement C) (2016) 197-203.

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Superfamily: Significant Evidence for Glycoside Hydrolase Signature Motifs, PLoS ONE 5(11) (2010)

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chitinase activity accompanied by quaternary structural change, J. Biol. Chem. 282(37) (2007) 27459-

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Foregut in Urechis unicinctus, Appl. Microsc. 28 (1998).

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[56] H.R. Ibrahim, T. Matsuzaki, T. Aoki, Genetic evidence that antibacterial activity of lysozyme is

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independent of its catalytic function, FEBS Lett. 506(1) (2001) 27-32.

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[57] B. Masschalck, D. Deckers, C.W. Michiels, Lytic and nonlytic mechanism of inactivation of

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gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure, J. Food Prot.

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65(12) (2002) 1916-1923.

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[58] W. Vollmer, Structural variation in the glycan strands of bacterial peptidoglycan, FEMS

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Microbiol. Rev. 32(2) (2008) 287-306.

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Methods Mol. Biol. (Clifton, N.J.) 1268 (2015) 43-66.

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Pharmacol. Rev. 55(1) (2003) 27-55.

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Table 1. Designations and nucleotide sequences of the primers used in this study.

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Table 2. AMP prediction chart for the fragments of the purified protein, Uu-ilys.

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ACCEPTED MANUSCRIPT Figure legends

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Fig. 1. Purification of the antibacterial material from the nephridia extract of Urechis unicinctus.

651

The fractions eluted between 4 to 10 min with citrate buffer (pH 5.6) on a cation-exchange column,

652

SP-5PW, showed strong antibacterial activity (A, red bar). The antibacterial activity against B. subtilis

653

is shown in a box. These fractions were separated further using a reverse phased column, CAPCELL-

654

PAK C18, and the fractions eluted from 25 to 32% acetonitrile/0.1% TFA had strong antibacterial

655

activity (B, red bar and box). Then, an anion-exchange column, Mono Q HR 5/5, was used to separate

656

antibacterial fractions, which were eluted between 1 to 6 min with Tris-HCl buffer (pH 8.0) (C, red

657

bar). These fractions were separated using CAPCELL-PAK C18 and an antibacterial fraction was

658

pooled for the final purification step (D, red bar).

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Fig. 2. Isolation of an antibacterial material and determination of its primary structure. An

660

antibacterial peak, which exhibited potent antibacterial activity against B. subtilis, was eluted between

661

26-27% acetonitrile/0.1% TFA on CAPCELL-PAK C18 (A, red arrow and box). An approximate

662

molecular weight of the isolated peak was investigated through SDS-PAGE and the isolated material

663

migrated to 14 kDa (B, red arrow). The molecular weight of the isolated protein was measured using

664

LC-MS, which gave [M+H]+ of the material as 13,295.1 Da (C). N-terminal AA sequences of the

665

isolated protein was obtained through automated Edman degradation, which gave a partial AA

666

sequence of 30 AAs (D). Residues marked with ‘X’ were residues that were not determined and were

667

predicted to be cysteines. The homology search through NCBI BLAST using the acquired partial AA

668

sequence revealed that the isolated protein was highly similar to i-type lysozymes described in other

669

annelid species: 66% identities with E. andrei (gbㅣABC68610.1ㅣ) and E. fetida (gbㅣAGJ83864.1ㅣ)

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and 62% with H. medicinalis (gbㅣAAA96144.1ㅣ) (D). The isolated protein was, then, designated

671

Urechis unicinctus-invertebrate type lysozyme (Uu-ilys).

672

Fig. 3. U. unicinctus-invertebrate type lysozyme (Uu-ilys) precursor. The DNA sequence

673

(lowercase, 714 bases) encoding Uu-ilys precursor (uppercase, 160 AAs). The predicted signal

674

peptide (18 AAs), pro-sequence (20 AAs), and the mature Uu-ilys (122 AAs) are shown in blue, black,

675

and red, respectively. The positions of the stop codon (asterisk) and the polyadenylation consensus

676

sequence (aataaa, underlined) are shown in the sequence.

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Fig. 4. Uu-ilys-precursor-transcript expression levels in five different tissues of U. unicinctus.

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Relative expression levels of Uu-ilys transcripts in each tissue normalized against β-actin levels. Data

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ACCEPTED MANUSCRIPT represent the mean ± standard deviation (n = 3) denoted by statistically significant differences (a, b, c,

680

and d; p < 0.05) between tissues as determined by one-way ANOVA, followed by Bonferroni

681

multiple-range test.

682

Fig. 5. Recombinant Uu-ilys (rUu-ilys) production using the pET28a-Uu-ilys construct in

683

Escherichia coli BL21(DE3) cells. Schematic representation of the pET28a-Uu-ilys expression

684

vector (A). His-tagged rUu-ilys was overexpressed in 0.25 mM IPTG at 37 °C for 6 h as insoluble

685

protein, which migrated to approx. 18 kDa (B). The affinity purified and dialyzed rUu-ilys was

686

compared with its native form on SDS-PAGE, in which rUu-ilys migrated less than the native Uu-ilys

687

on a 15% polyacrylamide gel (C).

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Fig. 6. Lysozyme and antibacterial activity of Uu-ilys. The lysozyme activity of the native (Uu-

689

ilys) and the recombinant (rUu-ilys) lysozymes, compared with the activity of HEWL, are shown in %

690

lysis of M. lydeikticus measured with absrobance at 450 nm (A). Among the nine bacterial strains

691

used in the antibacterial activity assay, Uu-ilys, rUu-ilys, and HEWL (100 µg/ml) showed the most

692

powerful antibacterial activity against two gram positive (B. subtilis and M. luteus) and two gram

693

negative bacteria (S. enterica and E. coli) (B). The MEC (µg/ml) of Uu-ilys, rUu-ilys, and HEWL

694

against the four most susceptible strains were measured (C). The experiments were conducted in

695

triplicate and averaged.

696

Fig. 7. Multiple sequence alignment and phylogenetic analysis of Uu-ilys with other i-type

697

lysozymes. Multiple sequence alignment of Uu-ilys and seven i-type lysozymes from Hirudo

698

medicinalis (phylum Annelida), Ruditapes philippinarum (phylum Mollusca) Caenorhabditis elegans

699

(phylum Nematoda), Drosophila melanogaster (phylum Arthropoda), Suberites domuncula (phylum

700

Porifera), Apostichopus japonicus (phylum Echinodermata), and Branchiostoma japonicum (phylum

701

Chordata) (A). The ribbon diagram represents the secondary structure of Vp-ilys from V.

702

philippinarum (below aligned sequences, A). Phylogenetic tree was built with the protein sequences

703

of several i-type lysozymes using ML method to evaluate the relationships among the i-type

704

lysozymes (B).

705

Fig. 8. Molecular model and prediction of AMP regions in Uu-ilys. The 3D model was constructed

706

using Vpi-ilys from V. pillippinarum (2DQA.1A) as a template (A). The glycoside hydrolase

707

structural motif (α-helix 1 and β-strands 2 and 3) and the catalytic residues responsible for

708

muramidase activity (Glu14 and Asp26) and isopeptidase activity (Ser62 and His92) are shown in orange,

709

red, and blue, respectively. Seven roughly fragmented regions reflecting the secondary structural

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ACCEPTED MANUSCRIPT features: α1, β1-3, α2, α3-4, α5, α6, α5-6 (B). Positions, lengths, and parameters considered for

711

antibacterial activity of the fragments are shown in Table 2. Helical wheel projections for α-helices 5

712

and 6 in α5-6 fragment, which was considered most likely to exert antibacterial activity (C, D).

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ACCEPTED MANUSCRIPT Primer Sequence (5’→3’)

Usage

Deg-Fw1 GCNTGYATHTGYAAYGTNGARGG Deg-nested-Fw2 TGYAAYGTNGARGGNTGYGA RACE PCR GSP-Rv1 CACAGCTTAATGAGCCGCG

Uu-ilys-BamHI-Fw ACGTGGATCCGCGATTTCCAACAACTGT

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GSP-Rv2 GGTCCATTCGGCACTTGC

Uu-ilys qPCR-F TTGTGCCAAGCAGATGGTCT Uu-ilys qPCR-R CCCCCTAGGACCTCCATTGT β-actin qPCR-F CCCATCTACGAGGGATACGC

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β-actin qPCR-R CCTTGATGTCACGGACGATT

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Uu-ilys-XhoI-Rv GCTTCTCGAGGCATCTCCCGCCAAACGAGG

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Recombinant production

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1

APD

APD

APD

APD

APD 1.29

0.670

NAMP

2.49

0.560

NAMP

1.79

1.67

0.123

NAMP

0.54

0.79

0.773

NAMP

39-59

21

47

-1

0.257

4.89

β1-3

60-76

17

35

3

-0.576

3.43

α2

77-89

13

23

-1

0.908

α3-4

90-108

19

57

2

0.211

α5

124-139 16

18

1

-1.375

5.93

2.79

0.197

AMP

α6

140-160 21

42

2

-0.414

2.61

2.34

0.730

NAMP

α5-6

124-160 37

32

3

8.54

2.53

0.643

AMP

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AMP probability AMP (Random prediction Forest classfier) CAMP DBAASP

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Fragment Position

Wimley-White Boman Total Net Index hydrophobicity GRAVY hydrophobicity charge (Kcal/mol) (Kcal/mol) ratio (%)

1

Helical Formation APD Y even # of cysteines (4) Y even # of cysteines (2) N Y 5 hydrophoibic residue on the same surface N Y 4 hydrophoibic residue on the same surface Y even # of cysteines (4)

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ACCEPTED MANUSCRIPT Highlights ▶ An invertebrate-type lysozyme, Uu-ilys, was isolated from nephridia of a marine annelid, Urechis unicinctus.

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▶ Uu-ilys exhibited muramidase and antibacterial activities that was comparable to activities of hen egg white lysozyme.

▶ Uu-ilys is involved in the immune defense in nephridia of U. unicinctus.

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▶ Recombinant Uu-ilys exerted antibacterial activity by non-enzymatic antibacterial mechanism.

1